Multi-component inhibitors of nucleic acid polymerases

ABSTRACT

The present invention provides multi-component inhibitors of nucleic acid polymerases, methods of making, and methods of using same. One component of the multi-component inhibitor is a molecule that binds to a polymerase (i.e., a polymerase-binding molecule (PBM)), but does not thereby substantially inhibit its polymerase activity. Another component is a molecule or complex of molecules that binds to a PBM (i.e., a PBM-binding molecule). The combination of the PBM and PBM-binding molecule/complex substantially inhibits polymerase activity. The disclosed multi-component inhibitors are useful for DNA sequencing, nucleic acid amplification, cloning and synthesis, and the like.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This present invention relates to compositions and methods forinhibiting nucleic acid polymerases. These compositions and methods maybe used for nucleic acid synthesis, amplification, sequencing andcloning.

2. Related Art

Nucleic acid polymerases (“polymerases”) are enzymes that catalyze thesynthesis of nucleic acid molecules that are complementary to a nucleicacid template. Template-directed nucleic acid synthesis is an importantaspect of many molecular biology research and diagnostic techniques andassays. Such techniques and assays typically involve extension of anucleic acid primer designed to hybridize to a specific region of thetemplate.

The yield and homogeneity of primer extension products made bypolymerases can be adversely affected by “mispriming,” i.e.,hybridization of primers to inappropriate regions of the template, or tonon-template nucleic acids. Extension of misprimed nucleic acids canproduce high background and obscure detection of properly primed primerextension products. In addition, diversion of nucleic acid synthesisreaction constituents to extend misprimed nucleic acids can reduce theyield of properly primed primer extension products, reducing thesensitivity of detection. The yield of primer extension products alsocan be adversely affected by template or primer degradation (e.g., by anuclease activity of a polymerase). Mispriming and template or primerdegradation can occur, e.g., when nucleic acid synthesis mixturescontaining template, primers and polymerase are maintained attemperatures associated with manufacture, shipping, storage or bench topassembly of such mixtures.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods and materials for synthesizing nucleicacids. The methods and materials of the invention can enhance the yieldand/or homogeneity of primer extension products made by polymerases.

One embodiment of the present invention is a composition comprising anucleic acid polymerase; a polymerase-binding molecule (PBM) that bindsto the polymerase and does not substantially inhibit the polymeraseactivity of the polymerase; and a PBM-binding molecule or complex ofmolecules that binds to the PBM such that binding of the PBM andPBM-binding molecule or complex together substantially inhibits thepolymerase activity of the polymerase. In one aspect of this embodiment,the PBM is an antibody (PBA). The antibody may be a monoclonal antibody.In another aspect, the PBM-binding molecule/complex is an antibody,protein G, protein A, a derivatized antibody, a derivatized protein G, aderivatized protein A, IgG and a derivatized protein G, sir22, sib A ora complex comprising any one of the foregoing. In one embodiment, thederivatized PBM-binding molecule or complex of molecules comprises adetectable label, protein or polymer. The detectable label may berhodamine, biotin, fluorescein, horseradish peroxidase, alkalinephosphatase or AlexaFluor488. The protein may be horseradish peroxidase,alkaline phosphatase or albumin. The polymer may be a polyethyleneglycol, a polyoxyethylene, a polyoxypropylene or apolyoxyethylene/polyoxyethylene copolymer. In one embodiment, theantibody is (a) IgA; (b) IgG; (c) IgM; (d) IgD; (e) IgE; (f) IgY; (g) afragment of any of (a)-(f); (h) a derivative of any of (a)-(f); (i) aderivatized fragment of any of (a)-(h); and (j) a complex of any of(a)-(i). In another embodiment, the PBM-binding molecule/complex is aderivatized or underivatized monoclonal antibody, polyclonal antibody,Fc antibody fragment, chimeric antibody or recombinant antibody. In yetanother embodiment, the derivatized antibody is derivatized byattachment of a detectable label, protein or polymer. In one aspect ofthis embodiment, the detectable label is rhodamine, biotin, fluorescein,horseradish peroxidase, alkaline phosphatase or AlexaFluor488. Inanother aspect of this embodiment, the protein is horseradishperoxidase, alkaline phosphatase or albumin. In yet another aspect ofthis embodiment, the polymer is a polyethylene glycol, apolyoxyethylene, a polyoxypropylene or a polyoxyethylene/polyoxyethylenecopolymer. In another embodiment, the PBM-binding molecule or complex ofmolecules is a goat anti-mouse IgG antibody coupled to horseradishperoxidase; a complex of a goat anti-mouse IgG antibody and proteinG-horseradish peroxidase; protein G-AlexaFluor488; a goat anti-mouse IgGantibody; a complex of streptavidin and an antibody to mouse IgG coupledto biotin; and Protein G. The nucleic acid polymerase may be aDNA-dependent DNA polymerase of an RNA-dependent DNA polymerase. In oneembodiment, the polymerase is thermolabile. In another embodiment, thepolymerase is thermostable. The polymerase may be Thermus aquaticus(Taq), Thermus thermophilus (Tth), Thermus filiformis (Tfi), Thermusflavus (Tfl), Pyrococcus furiosus (Pfu), Thermococcus litoralis (Tli),Thermococcus zilligi (Tzi), Thermatoga neopolitana (Tne), Thermatogamaritime (Tma), VENT®, DEEPVENT®, THERMOSCRIPT®, SUPERSCRIPT I®,SUPERSCRIPT II®, SUPERSCRIPT III® polymerase and a mutant of any of theabove. In one embodiment, the polymerase is recombinant.

The present invention also provides a composition comprising athermostable nucleic acid polymerase; a polymerase-binding antibody(PBA) that binds to the polymerase and does not substantially inhibitthe polymerase activity of the polymerase; and a PBA-binding molecule orcomplex of molecules that binds to the PBA, such that binding of the PBAand PBA-binding molecule or complex together substantially inhibits thepolymerase activity of the polymerase at a temperature less than about40° C., and such that the binding of the PBA and PBA-binding molecule orcomplex together does not substantially inhibit the polymerase activityof the polymerase at a temperature greater than about 40° C. The nucleicacid polymerase may be a DNA-dependent DNA polymerase of anRNA-dependent DNA polymerase. In one embodiment, the polymerase isthermolabile. In another embodiment, the polymerase is thermostable. Thepolymerase may be Thermus aquaticus (Taq), Thermus thermophilus (Tth),Thermus filiformis (Tfi), Thermus flavus (Tfl), Pyrococcus furiosus(Pfu), Thermococcus litoralis (Tli), Thermococcus zilligi (Tzi),Thermatoga neopolitana (Tne), Thermatoga maritime (Tma), Vent®,DeepVent®, Thermoscript®, Superscript I®, Superscript II®, SuperscriptIII® polymerase and a mutant of any of the above. In one embodiment, thepolymerase is recombinant.

There is also provided a method of inhibiting the polymeras activity ofa nucleic acid polymerase comprising contacting the polymerase with aPBM and PBM-binding molecule or complex of molecules, wherein thebinding of the PBM does not substantially inhibit the polymeraseactivity of the polymerase, and in which the binding of the PBM and thePBM-binding molecule or complex of molecules together substantiallyinhibits the polymerase activity of the polymerase. The inhibition maybe irreversible, The inhibition may also be reversible by heating to atemperature of at least about 40° C. In one embodiment, the PBM is aPBA. In another embodiment, the PBM-binding molecule is an antibody. Thenucleic acid polymerase may be a DNA-dependent DNA polymerase or anRNA-dependent DNA polymerase. The polymerase may be Thermus aquaticus(Taq), Thermus thermophilus (Tth), Thermus filiformis (Tfi), Thermusflavus (Tfl), Pyrococcus furiosus (Pfu), Thermococcus litoralis (Tli),Thermococcus zilligi (Tzi), Thermatoga neopolitana (Tne), Thermatogamaritime (Tma), Vent®, DeepVent®, Thermoscript®, Superscript I®,Superscript II®, Superscript III® polymerase and a mutant of any of theabove. In one embodiment, the polymerase is recombinant.

The present invention also provides a method for synthesizing a nucleicacid molecule, comprising contacting a template nucleic acid with acomposition comprising a thermostable nucleic acid polymerase, one ormore nucleoside and/or deoxynucleoside triphosphates and an inhibitor ofsaid polymerase, in which the inhibitor comprises a PBM that does notsubstantially inhibit the polymerase activity of the polymerase; and aPBM-binding molecule or complex of molecules, such that the combinationof the PBM and the PBM-binding molecule/complex substantially inhibitsthe polymerase activity of the polymerase; and bringing the resultingmixture to a temperature sufficient to inactivate the inhibitor, butthat does not inactivate the polymerase. In one embodiment, the PBM is aPBA. The PBM-binding molecule may be an antibody. In one embodiment, theinhibition is irreversible. In another embodiment, the inhibition isreversible by heating to a temperature of at least about 40° C. Thenucleic acid polymerase may be a DNA-dependent DNA polymerase or anRNA-dependent DNA polymerase. The polymerase may be Thermus aquaticus(Taq), Thermus thermophilus (Tth), Thermus filiformis (Tfi), Thermusflavus (Tfl), Pyrococcus furiosus (Pfu), Thermococcus litoralis (Tli),Thermococcus zilligi (Tzi), Thermatoga neopolitana (Tne), Thermatogamaritime (Tma), Vent®, DeepVent®, Thermoscript®, Superscript I®,Superscript II®, Superscript III® polymerase and a mutant of any of theabove. In one embodiment, the polymerase is recombinant.

Other preferred embodiments will be apparent to one of ordinary skill inlight of the drawings, description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Bar graph showing the effect of multicomponent inhibitors onSSIII activity. SSIII activity is depicted in the presence of eachspecified anti-SSIII primary antibody alone (light bars) or withanti-SSIII primary antibody plus goat anti-mouse-IgG-horse radishperoxidase (dark bars). All other activities are normalized toreaction 1. Column 1: No primary mAb or inhibitor components. SSIIIactivity set to 100% by definition. Columns 2-9: Anti-SSIII mAb clone #4in molar ratios of SSIII: mAb clone #4 of 1:10 to 2:1. Columns 10-15:Anti-SSIII mAb clone #2 in molar ratios of SSIII: mAb clone #2 of 1:10to 2:1. Columns 16-17: Anti-SSIII mAb clones #2 and #4 in molar ratiosof SSIII: mAb clones #2 and #4 of 1:2. Column 18: Anti-SSIII mAb clones#2 and #4 in molar ratios of SSIII: mAb clones #2 and #4 of 1:2, wherethe anti-SSIII mAbs were heated to 96° for 7 minutes prior to adding tothe SSIII.

FIG. 2. Bar graph showing SSIII activity in the presence of differentanti-SSIII primary mAb, each used as part of a multicomponent inhibitor.SSIII activity in reactions is normalized to that in Column 1. Column 1:No primary mAb or inhibitor components. Activity set to 100% bydefinition. Column 2: Anti-ThermoScript mAb DE11 withgoat-anti-mouse-IgG-horse radish peroxidase. Columns 3-6: Decreasingamounts of anti-SSIII mAb with constant amount ofgoat-anti-mouse-IgG-horse radish peroxidase. Columns 7-10: Four separateanti-SSIII clones with goat-anti-mouse-IgG-horse radish peroxidase.Columns 11-13: Constant amount of anti-SSIII mAb clone #4 withdecreasing amounts of goat-anti-mouse-IgG-horse radish peroxidase.

FIG. 3. Bar graph showing the effect of anti-SSIII primary mAb andseveral different inhibitor components on SSIII activity. SSIII activityis depicted in the presence of each specified inhibitor component eitherwithout anti-SSIII primary mAb (light bars), or with anti-SSIII primarymAb (dark bars). SSIII activity in reactions is normalized to that inColumn 1. Column 1: No primary mAb or inhibitor components. Column 2:Goat-anti-mouse-IgG-Horse radish peroxidase. Columns 3-4: Concavalin A(ConA). Columns 5-6: Protein G-AlexaFluor 488. Columns 7-8:Goat-anti-mouse-IgG-biotin+Streptavidin. Column 9: Goat anti-mouse-IgG.Column 10: Goat anti-mouse-IgG+ConA. Column 11: Goatanti-mouse-IgG+Protein G-AlexaFluor 488.

FIG. 4. Bar graph showing the effect of multicomponent inhibitors atdifferent temperatures on SSIII activity. Light bars depict polymeraseactivity at 270 and dark bars depict activity at 55°. SSIII polymeraseactivity in each reaction is normalized to that in Column 1. Columns1-2: No primary mAb or inhibitor components. SSIII activity at 27° and55° are each independently set to 100% by definition. Columns 3-4:Anti-ThermoScript primary mAb with Gmix (Goat-anti-mouse-IgG+ProteinG-Alexafluor 488). Columns 5-6: Anti-SSIII primary mAb withgoat-anti-mouse-IgG-horse radish peroxidase. Columns 7-8: Anti-SSIIIprimary mAb with Gmix.

FIG. 5. Bar graph showing the effect of multicomponent inhibitors onThermococcus zilligi (Tzi) thermostable DNA polymerase activity. Tziactivity is depicted in the presence of each specified component withthe addition of buffer only (light bars) or Gmix (goatanti-mouse-IgG+Protein G-AlexaFluor 488, dark bars). Tzi polymeraseactivity in each reaction is normalized to that in Column 1. Column 1:No primary anti-Tzi antibody or inhibitor components. Set to 100% bydefinition. Column 2: Gmix only. Columns 3-4: Primary anti-Tzi antibodyclone 8C9.2. Columns 5-6: Primary anti-Tzi antibody clone 3B11.2.Columns 7-8: Primary anti-Tzi antibody clone 8F3.2. Columns 9-10:Primary anti-Tzi antibody clone 9G2.2. Columns 11-12: Primary anti-Tziantibody clone 802.2. Columns 13-14: Primary anti-Tzi antibody clone10F7.3.

FIG. 6. Bar graph showing the effect of multicomponent inhibitors atdifferent temperatures on Tzi activity. Light bars depict Tzi activityat 37° C. and dark bars depict activity at 74° C. Tzi polymeraseactivity in reactions is normalized to that in Column 1. Columns 1-2: Noprimary anti-Tzi mAb or inhibitor components. Tzi activity at 37° and74° are each independently set to 100% by definition. Columns 3-4: Gmix(goat-anti-mouse-IgG+Protein G-AlexaFluor 488) without primary anti-TzimAb. Columns 5-6: Primary anti-Tzi mAb clone 1G11.3 with Gmix.

FIG. 7. Line graph showing Tzi polymerase activity in the presence ofincreasing concentrations of anti-Tzi polymerase monoclonal antibodiesin the presence of absence of secondary inhibitors.

FIG. 8: Line graph showing RT-41 polymerase activity in the presence ofconstant concentration of anti-RT-41 mAb #10 and increasingconcentrations of anti-RT-41 mAb #5 with or without rabbit anti-mouseIgG secondary antibody.

FIG. 9: Line graph showing Taq polymerase activity in the presence ofconstant concentration of anti-Taq mAb #10 and increasing concentrationsof anti-Taq mAb #5 with or without rabbit anti-mouse IgG secondaryantibody.

FIG. 10: Line graph showing Tzi polymerase activity in the presence ofincreasing concentrations of rabbit-anti-mouse IgG in the presence orabsence of anti-Tzi polymerase monoclonal antibodies.

FIG. 11: Line graph showing Tzi polymerase activity after 2 minutes ofpreincubation at various temperatures in the presence of absence ofanti-Tzi polymerase monoclonal antibody and secondary inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

General Definitions

“A,” “An” and “One” include both the singular and plural, unlessotherwise indicated or unless it is clear from the context in which theterm is used that one or the other is intended.

“About” refers to a value that is within plus or minus 10% of areference value. For example, a value of about 50° C. would encompass arange of values between 45° C. and 55° C.

“Amplification” refers to any in vitro method for increasing the numberof copies of a nucleotide sequence with the use of a polymerase.Amplification results in the incorporation of nucleotides into a nucleicacid (e.g., DNA) molecule or primer thereby forming a new nucleic acidmolecule complementary to the nucleic acid template. Typically, thetemplate and newly formed nucleic acid molecule can be used as templatesto synthesize additional nucleic acid molecules. As used herein, oneamplification reaction may consist of many rounds of nucleic acidsynthesis. Amplification reactions include, for example, polymerasechain reactions (PCR). One PCR-type amplification may consist of 5 to100 or more rounds of denaturation and synthesis of a nucleic acidmolecule.

“Antibody” refers to molecule(s) that are capable of binding an epitopeor antigenic determinant. The term is meant to include whole antibodiesand antigen-binding fragments thereof, including Fab, Fab′ and F(ab′)2,Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linkedFvs (sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies can be from any animal origin including birds such aschicken, and mammals such as human, murine, rat, rabbit, goat, guineapig, sheep, cow, camel and horse. The term “antibodies” also includesgenetically prepared equivalents thereof, and chemically or geneticallyprepared fragments of antibodies (such as Fab fragments), recombinantantibodies, chimeric antibodies, monoclonal, polyclonal, affinitypurified polyclonal, and the like. Antibodies and fragments thereof, canbe used singly or in mixtures in the practice of this invention.

“Bound” means to be coupled via covalent or non-covalent interactions.Covalent binding can occur via chemically coupling and the formation of,e.g., ester, ether, phosphoester, thioester, thioether, urethane, amide,amine, peptide, imide, hydrazone, hydrazide, carbon-sulfur,carbon-phosphorus, and like bonds. Non-covalent binding can occur via,e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.

“Exonuclease activity” relates to enzymatic activity resulting in theremoval of nucleotides from a polynucleotide, in either the 3′-to-5′direction (“3′-to-5′ exonuclease activity”) or the 5′-to-3′ direction(“5′-to-3′ exonuclease activity”). A polymerase may exhibit either orboth 3′-to-5′ and 5′-to-3′ exonuclease activity. Modified or recombinantpolymerases are available in which either or both have beensubstantially reduced or eliminated.

“Hybridization” and “hybridizing” refer to the pairing of twocomplementary single-stranded nucleic acid molecules (RNA and/or DNA) togive a double-stranded molecule. Two nucleic acid molecules may behybridized, although the base pairing is not completely complementary.Accordingly, mismatched bases do not prevent hybridization of twonucleic acid molecules provided that appropriate conditions, well knownin the art, are used. Hybridization refers in some contexts to pairingof an oligonucleotide with a DNA template molecule.

“Inactivated” refers to a reduction of a specified property or activityto less than 10%, 7.5%, 5%, 2.5%, 1%, 0.5% or 0.1% of its originalproperty or activity including the polymerase activity of a polymeraseor the inhibitory activity of an inhibitor.

“Nucleotide” refers to a base-sugar-phosphate combination. Nucleotidesare monomeric units of a nucleic acid (DNA and RNA). The term nucleotideincludes deoxyribonucleoside triphosphates (“dNTPs”), such as dATP,dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivativesinclude, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP. The termnucleotide also includes dideoxyribonucleoside triphosphates (“ddNTPs”)and their derivatives, such as ddATP, ddCTP, ddGTP, ddITP, and ddTTP.The term nucleotide also includes ribonucleoside triphosphates (rNTPs)such as rATP, rCTP, rITP, rUTP, rGTP, rTTP and their derivatives, whichare analogous to the above-described dNTPs and ddNTPs except that therNTPs comprise ribose instead of deoxyribose or dideoxyribose in theirsugar-phosphate backbone. The term “NTP” is more general and mayencompass rNTP, dNTP, ddNTP or nucleotide analogs. A “nucleotide” may beunlabeled or detectably labeled by well known techniques. Detectablelabels include, for example, radioactive isotopes, fluorescent labels,chemiluminescent labels, bioluminescent labels and enzyme labels.

“Nucleic acid” and “Nucleic acid molecule” refer to a series ofcontiguous nucleotides which may encode a full-length polypeptide or afragment of any length thereof, or which may be non-coding.

“Nucleic acid polymerase” and “Polymerase” refer to any polypeptide,protein or enzyme with nucleic acid polymerase activity.

“Oligonucleotide” refers to a synthetic or natural molecule comprising acovalently linked sequence of nucleotides which are joined by aphosphodiester bond between the 3′ position of the pentose of onenucleotide and the 5′ position of the pentose of the adjacentnucleotide.

“Polymerase activity” is an enzymatic activity, whereby a polymerasesynthesizes polynucleotides in the 5′ to 3′ direction by addition of anew nucleotide to the 3′ end of a the previous nucleotide, according toan RNA or DNA template that directs the synthesis of the polynucleotide.For example, a DNA polymerase can synthesize the formation of a DNAmolecule complementary to a single-stranded DNA or RNA template byextending a primer in the 5′-to-3′ direction. Polymerases includeDNA-dependant DNA polymerases; DNA-dependant RNA polymerases, also knownas transcriptases; RNA-dependant DNA polymerases, also known as reversetranscriptases; and, more often seen in certain viruses, RNA-dependantRNA polymerases. A given polymerase enzyme may have more than onepolymerase activity. For example, some DNA-dependent DNA polymerases,such as Taq, also exhibit reverse transcriptase polymerase activity.

“Polypeptide,” “Peptide” and “Protein” refer to series of contiguousamino acids, of any length.

“Primer” refers to a single-stranded oligonucleotide that is extended bycovalent bonding of nucleotide monomers during amplification orpolymerization of a DNA molecule.

“Stable” and “Stability” refer to the retention by an enzyme of at leastabout 70%, at least about 80%, or at least about 90%, of the originalenzymatic activity (in units) after the enzyme or composition containingthe enzyme has been subjected to a condition which might otherwise haveresulted in loss of activity for an enzyme that was not stable. Labileis the opposite of stable.

“Substantially pure” means that the desired purified molecule such as aprotein or nucleic acid is essentially free from contaminants typicallyassociated with the desired molecule. Contaminating components includecompounds or molecules that may interfere with the inhibitory orsynthesis reactions of the invention, and/or that degrade or digest themolecules of the invention and/or that degrade or digest the synthesizedor amplified nucleic acid molecules produced by the methods of theinvention.

“Template” refers to a double-stranded or single-stranded nucleic acidmolecule that is to be amplified, synthesized or sequenced. In the caseof a double-stranded RNA or DNA molecule, denaturation of its strands toform a first and a second strand is performed before these molecules maybe amplified, synthesized or sequenced. A primer complementary to aportion of a template is hybridized to the template under appropriateconditions and a polymerase of the invention may then synthesize anucleic acid molecule complementary to the template or a portionthereof. The newly synthesized DNA molecule, according to the invention,may be equal or shorter in length than the original DNA template.Mismatch incorporation or strand slippage during the synthesis orextension of the newly synthesized DNA molecule may result in one or anumber of mismatched base pairs. Thus, the synthesized nucleic acidmolecule need not be exactly complementary to the template.

“Thermostable” refers to an enzyme (such as a polypeptide havingpolymerase activity) that is resistant to inactivation by heat. A“thermostable” enzyme is in contrast to a “thermolabile” polymerase,which can be inactivated by heat treatment. Thermolabile proteins can beinactivated at physiological temperatures, and can be categorized asmesothermostable (inactivation at about 45° C. to 65° C.), and athermostable (inactivation greater than about 65° C.). For example, theactivities of the thermolabile T5 and T7 DNA polymerases can be totallyinactivated by exposing the enzymes to a temperature of about 90° C. forabout 30 seconds. A thermostable polymerase activity is more resistantto heat inactivation than a thermolabile polymerase. However, athermostable polymerase does not mean to refer to an enzyme that istotally resistant to heat inactivation; thus heat treatment may reducethe polymerase activity to some extent. A thermostable polymerasetypically will also have a higher optimum temperature than thermolabileDNA polymerases.

“Unit” refers to the activity of an enzyme. When referring to athermostable polymerase (e.g., Taq and Pfx), one unit of activity is theamount of enzyme that will incorporate 10 nanomoles of NTPs intoacid-insoluble material (i.e., DNA or RNA) in 30 minutes under standardprimed DNA synthesis conditions at 74° C. When referring to reversetranscriptases (e.g. SuperScript III), one unit is defined as the amountof enzyme which incorporates 1 nmole of dTTP into acid insolublematerial in 10 min at 37° C.

“Working concentration” refers to the concentration of a reagent that isat or near the optimal concentration used in a solution to perform aparticular function (such as amplification or digestion of a nucleicacid molecule). The working concentration of a reagent is also describedequivalently as a “1× concentration” or a “1× solution” (if the reagentis in solution) of the reagent. Accordingly, higher concentrations ofthe reagent may also be described based on the working concentration;for example, a “2× concentration” or a “2× solution” of a reagent isdefined as a concentration or solution that is twice as high as theworking concentration of the reagent; a “5× concentration” or a “5×solution” is five times as high as the working concentration of thereagent; and so on.

Compositions

The invention provides compositions that include a polymerase-bindingmolecule (PBM) and a PBM-binding molecule or complex, where binding ofthe PBM to a polymerase does not substantially inhibit the polymeraseactivity of the polymerase, but where binding of the PBM and PBM-bindingmolecule/complex together substantially inhibit the polymerase activityof the polymerase. The PBM and PBM-binding molecule/complex may alsoinhibit the 3′-5′ exonuclease activity, 5′-3′ exonuclease activityand/or RNase H activity of a polymerase.

The PBM does not substantially inhibit the polymerase activity of apolymerase in the absence of the PBM-binding molecule/complex.Accordingly, a polymerase bound by a PBM in the absence of a PBM-bindingmolecule/complex exhibits at least about 70%, 75%, 80%, 85%, 90%, 95%,97.5%, 99%, 99.75%, 100% or >100% of the polymerase activity observed inthe absence of the PBM.

Binding of a PBM together with a PBM-binding molecule/complexsubstantially inhibits the polymerase activity of a polymerase.Accordingly, a polymerase bound by a PBM in the presence of aPBM-binding molecule/complex exhibits less than about 30%, 25%, 20%,15%, 10%, 5%, 2.5% 1% or 0.25% of the polymerase activity observedeither in the absence of both the PBM and PBM-binding molecule/complex,or in the presence of the PBM but in the absence of the PBM-bindingmolecule/complex.

Polymerase inhibition by a PBM and PBM-binding molecule/complex may beirreversible or reversible. Inhibition of polymerase activity may bereversed by any means known in the art including, e.g., dilution,competition, physical or ionic disruption, or temperature change. Forexample, heating a composition containing a polymerase, PBM andPBM-binding molecule/complex may reverse polymerase inhibition. Suchheating typically involves a shift to a higher temperature that does notsubstantially reduce the polymerase's polymerase activity (e.g.,temperatures up to 40° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75°C., 80° C., 90° C., 95° C. or 99° C.). Such heating may be sufficient tocause denaturation of the PBM (e.g., antibody); denaturation of thePBM-binding molecule/complex; dissociation of the PBM from the nucleicacid polymerase; dissociation of the PBM-binding molecule/complex fromthe PBM; dissociation of a PBM-binding complex, or a combination ofthese effects.

Polymerase Binding Molecules (PBMs)

A PBM can be an antibody, antibody fragment, chemical compound, acid,antibiotic, heavy metal, metal chelator, nucleotide analog, sulfhydrylreagent, anionic detergent, polyanion, captan((N-[trichloromethyl]-thio)-4-cyclohexene-1,2-dicarboximide), acidicpolysaccharide or lectin.

A PBM can be specific for a DNA-dependent a DNA polymerase, aDNA-dependent RNA polymerase, a RNA-dependent DNA polymerase (reversetranscriptase) and/or a RNA-dependent RNA polymerase. Thus, compositionsin accord with the invention can include a PBM that binds a DNApolymerase such as Taq DNA polymerase, Tzi DNA polymerase, Tne DNApolymerase, Tma DNA polymerase, Pfu DNA polymerase, Tfl DNA polymerase,Tth DNA polymerase, Pwo DNA polymerase, Bst DNA polymerase, Bca DNApolymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, T7 DNApolymerase, T5 DNA polymerase, DNA polymerase III, Klenow fragment DNApolymerase, Stoffel fragment DNA polymerase, and/or mutants, fragmentsor derivatives thereof. Compositions in accord with the invention alsocan include a PBM that binds a polymerase having reverse transcriptaseactivity, such as an M-MLV reverse transcriptase, an RSV reversetranscriptase, an AMV reverse transcriptase, an RAV reversetranscriptase, an MAV reverse transcriptase, an HIV reversetranscriptase (any of which may be reduced in, substantially reduced in,or have no detectable RNase H activity) and/or mutants, fragments orderivatives thereof.

Compositions in accord with the invention can include a PBM that bindsto a thermolabile polymerase, and/or to a thermostable nucleic acidpolymerase, such as a Thermus aquaticus polymerase, a Thermusthermophilus polymerase, a Thermus filiformis polymerase, a Thermusflavus polymerase, a Pyrococcus furiosus polymerase, a Thermococcuslitoralis, Thermococcus zilligi or a Thermotoga species polymerase, or arecombinant variant thereof.

Certain exemplary compositions of the invention include antibody PBMs(“PBAs”). Accordingly, the invention provides compositions that includea polymerase-binding antibody (PBA) or derivative and a PBA-bindingmolecule/complex, where binding of the PBA or derivative to a polymerasedoes not substantially inhibit the polymerase activity of thepolymerase, but where binding of the PBA and PBA-bindingmolecule/complex together substantially inhibit the polymerase activityof the polymerase.

A PBA can be a monoclonal antibody, a polyclonal antibody, an Fcantibody fragment, a chimeric antibody or a recombinant or otherderivative thereof. A PBA can be an IgA antibody, an IgG antibody, anIgM antibody, an IgD antibody, an IgE antibody, an IgY antibody, an IgEantibody or fragment thereof. Specific monoclonal PBAs include:Anti-SSIII mAb clone #4, Anti-SSIII mAb clone #2, Primary anti-Tziantibody clone 8C9, Primary anti-Tzi antibody clone 3B11.2, Primaryanti-Tzi antibody clone 8F3.2, Primary anti-Tzi antibody clone 9G2.2,Primary anti-Tzi antibody clone 802.2, Primary anti-Tzi antibody clone10F7.3, Primary anti-Tzi mAb clone 1G11.3, anti-ThermoScript mAb DE11,anti-Rt41A mAb #5, anti-Rt41A mAb #10, anti-Taq mAb #5 and anti-Taq mAb#10.

PBM-Binding Molecules/Complexes

A PBM-binding molecule/complex can be any molecule/complex known in theart to be capable of binding a PBM.

When the PBM is an antibody (i.e., a PBA), the PBM-bindingmolecule/complex is capable of binding an antibody, and can be referredto as a PBA-binding molecule/complex. Many molecules/complexes capableof binding antibodies are known to those skilled in the art. Thus, aPBA-binding molecule/complex can include an antibody, an antibodyfragment (e.g., Fab fragment), protein G, protein A, a derivatizedantibody, a derivatized protein G, a derivatized protein A, protein H,ARP or a complex including any of the foregoing with another moiety.

In some embodiments, a PBA-binding molecule/complex comprises anantibody. The antibody may be monoclonal, polyclonal, chimeric, or an Fcfragment. The antibody may also be recombinant. In another embodiment,the antibody may be IgA, IgG, IgM, IgE, IgY, IgD or derivative thereof.A PBA-binding antibody may be from any species (including humans,monkeys, mice, rats, rabbits, horses, goats, sheep, cows, pigs,chickens, fish, etc.) and may bind to antibodies from any species(including humans, monkeys, mice, rats, rabbits, horses, goats, sheep,cows, pigs, chickens, fish, etc.). PBA-binding antibodies may bederivatized or underivatized monoclonal antibodies, derivatized orunderivatized polyclonal antibodies, derivatized or underivatized Fc orFab antibody fragments, derivatized or underivatized chimericantibodies, or derivatized or underivatized recombinant antibodies.PBA-binding antibodies can be IgA, IgG, IgM, IgD, IgE, IgY antibodies orfragments or derivatives thereof.

In some embodiments, a PBA-binding molecule/complex comprises abacterial immunoglobulin binding protein (IgBP) such as, e.g.,Staphylococcus aureus protein A, which binds to all immunoglobulinmolecules, and streptococcal protein G, which binds specifically to IgG.Other bacterial IgBPs are also available and may be used in thecompositions and methods described herein including, for example,Haemophilus somnus high molecular weight IgBPs, and Peptostreptococcusmagnus protein L, which binds immunoglobulin (Ig) light chains. Avariety of eukaryotic antibody-binding proteins may also be used, suchas Fc receptors on cells of the immune system. In addition, otherimmunoglobulin binding proteins may be used, including sir22 (Stenberget al., J. Biol. Chem. 269:13458-13464, 1994), sibA (Fagan et al.,Infect. Immun. 69:4851-4857, 2001) and ARP (U.S. Pat. No. 5,180,810;European Patent Application No. 0367890 A1).

PBM-binding molecules (including PBA-binding molecules such asantibodies) and molecules comprising PBM-binding complexes, may bederivatized with one or more molecules or moieties using well-knownprocedures, such as chemical coupling or recombinant DNA technology.Derivatization moieties include, e.g., detectable labels, signalinggroups, proteins, chemical groups, ligands, haptens or polymers. Adetectable label can be enzymatic, chemiluminescent, bioluminescent,radioactive or fluorescent. Exemplary detectable labels includerhodamine, biotin, fluorescein, horseradish peroxidase, alkalinephosphatase, and AlexaFluor488. Exemplary derivatization proteinsinclude horseradish peroxidase, alkaline phosphatase, protein G, andalbumin. Exemplary derivatization polymers include polyethylene glycol,polyoxyethylene, polyoxypropylene, and polyoxyethylene/polyoxypropylenecopolymer.

Derivatization typically alters the properties of a PBM-bindingmolecule. For example, large moieties including polymers, proteins andlarge chemical groups will increase the bulkiness of a PBM-bindingmolecule/complex. Attached moieties may also alter the net charge,solubility, ionic strength or other physical or chemical property of aPBM. Attached moieties may also serve as targets for other bindingmolecules.

PBM-binding molecules and PBM-binding complexes of molecules may be usedin the compositions and methods described herein. By way of example, aPBA-binding complex may include a PBA-binding antibody, to which isbound Protein G. Other exemplary PBA-binding complexes include anantibody coupled to horseradish peroxidase; an antibody-proteinG-horseradish peroxidase complex; a protein G-AlexaFluor488 complex; andan antibody-biotin-streptavidin complex. Yet another exemplaryPBA-binding complexes include a PBA-binding antibody derivatized with ahapten, which can bind an antibody against the hapten. In this manner,the number of antibodies, moieties and the like associated with a giventarget polymerase to be inhibited can be varied as desired.

Polymerases

Compositions of the invention may include one or more DNA and/or RNApolymerases. The polymerases may be thermolabile or thermostable. DNApolymerases include, but are not limited to, Thermus thermophilus (Tth)DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermococcuszilligi (Tzi), Thermotoga neopolitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNApolymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT™ DNApolymerase, Pyrococcus woosii (Pwo) DNA polymerase, Pyrococcus sp KOD2(KOD) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus acidocaldarius(Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase,Thermus flavus (Tfl/Tub) DNA polymerase, Thermus ruber (Tru) DNApolymerase, Thermus brockianus (DYNAZYME™) DNA polymerase,Methanobacterium thermoautotrophicum (Mth) DNA polymerase, amycobacterium DNA polymerase (e.g. Mtb, Mlep); and generally Pol I andPol III type polymerases.

Thermolabile Pol I and Pol III type nucleic acid polymerases and theirrespective Klenow fragments include, but are not limited to, those whichmay be isolated from organisms such as E. coli, H. influenzae, D.radiodurans, H. pylori, C. aurantiacus, R. prowazekii, T. pallidum,Synechocysis sp., B. subtilis, L. lactis, S. pneumoniae, M.tuberculosis, M. leprae and M. smegmatis bacteria; L5, phi-C31, T7, T3,T5, SP01, and SP02 bacteriophage; S. cerevisiae MIP-1 mitochondria;eukaryotes C. elegans, and D. melanogaster (Astatke, M. et al., 1998, J.Mol. Biol. 278, 147-165); and viral nucleic acid polymerases; and anymutants, variants and derivatives thereof.

Thermostable DNA polymerases that may be used in the methods andcompositions of the invention include Tfi; Tfl; Tzi; Tne; Tma; Pfu; Pwo;Vent®; KOD, Stoffel fragment, DeepVent®; Thermoscript®; Superscript I®;Superscript II®; Superscript III®; and mutants or variant thereof. Suchpolymerases are described, for example, in U.S. Pat. No. 5,436,149; U.S.Pat. No. 4,889,818; U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,079,352;U.S. Pat. No. 5,614,365; U.S. Pat. No. 5,374,553; U.S. Pat. No.5,270,179; U.S. Pat. No. 5,047,342; U.S. Pat. No. 5,512,462; WO92/06188; WO 92/06200; WO 96/10640; WO 97/09451; Barnes, W. M. Gene112:29-35 (1992); Lawyer, F. C., et al, PCR Meth. Appl. 2:275-287(1993); Flaman, J.-M, et al., Nucl. Acids Res. 22(15):3259-3260 (1994)).In one embodiment, DNA polymerase from Thermus Rt41A (herein called“Rt41A”), a species of Thermus filiformis, is used in the compositionsand methods described herein. The Rt41A nucleotide and protein sequencesare disclosed in SEQ ID NOS: 9 and 21, respectively, in WO03/025132 andcopending U.S. patent application Ser. No. 10/244,081, the entirecontents of which are incorporated herein by reference. Isolation andcharacterization of Tzi polymerase is described in copending U.S.Provisional Patent Application Ser. No. ______, entitled “DNA Polymerasefrom Thermococcus zilligi and Mutants Thereof” listing inventors Jun. E.Lee, Kyusung Park, Katherine R. Griffiths, Moreland D. Gibbs, and PeterL. Bergquist, filed on the same date as the present application, theentire contents of which are incorporated herein by reference.

RNA polymerases suitable for use in the compositions and methodsdescribed herein include any enzyme having RNA polymerase activity,including both DNA-dependent and RNA-dependant RNA polymerases. Moretypically, RNA polymerases used in the present invention will beDNA-dependent RNA polymerases, also known as reverse transcriptases.Transcriptases may be isolated from any source, including E. coli, T3,T5, SP-6, T7 and Xenopus, and mutants, variants and derivatives thereof.

Reverse transcriptases include any enzyme having reverse transcriptaseactivity. Such enzymes include, but are not limited to, retroviralreverse transcriptase, retrotransposon reverse transcriptase, hepatitisB reverse transcriptase, cauliflower mosaic virus reverse transcriptase,bacterial reverse transcriptase, Tth DNA polymerase, Taq DNA polymerase(Saiki, R. K., et al, Science 239:487-491 (1988); U.S. Pat. Nos.4,889,818 and 4,965,188; Shandilya et al., Extremophiles 8(3):243-251,2004), Tne DNA polymerase (WO 96/10640 and WO 97/09451), Tma DNApolymerase (U.S. Pat. No. 5,374,553) and mutants, variants orderivatives thereof (see, e.g., WO 97/09451 and WO 98/47912).

In one embodiment, reverse transcriptases include those that havereduced, substantially reduced or eliminated RNase H activity. By anenzyme “substantially reduced in RNase H activity” is meant that theenzyme has less than about 20%, 15%, 10%, 5%, or 2%, of the RNase Hactivity of the corresponding wild type or RNase H+ enzyme such as wildtype Moloney Murine Leukemia Virus (M-MLV), Avian Myeloblastosis Virus(AMV) or Rous Sarcoma Virus (RSV) reverse transcriptases. The RNase Hactivity of any enzyme may be determined by a variety of assays, such asthose described, for example, in U.S. Pat. No. 5,244,797, in Kotewicz,M. L., et al, Nucl. Acids Res. 16:265 (1988) and in Gerard, G. F., etal., FOCUS 14(5):91 (1992), the disclosures of all of which are fullyincorporated herein by reference. Polypeptides suitable for use in thecompositions and methods described herein include, but are not limitedto, M-MLV H⁻ reverse transcriptase, RSV H⁻ reverse transcriptase, AMV H⁻reverse transcriptase, RAV (Rous-associated virus) H⁻ reversetranscriptase, MAV (myeloblastosis-associated virus) H⁻ reversetranscriptase and HIV H⁻ reverse transcriptase (See U.S. Pat. No.5,244,797 and WO 98/47912), and superscript III. It will be understoodby one of ordinary skill, however, that any enzyme capable of producinga DNA molecule from a ribonucleic acid molecule (i.e., having reversetranscriptase activity) may be equivalently used in the compositions,methods and kits described herein, including those described in PCTWO03/025132, the entire disclosure of which is incorporated herein byreference.

The enzymes having polymerase may be obtained commercially, for examplefrom Invitrogen (Carlsbad, Calif.), Perkin-Elmer (Branchburg, N.J.), NewEngland BioLabs (Beverly, Mass.) or Boehringer Mannheim Biochemicals(Indianapolis, Ind.). Alternatively, polymerases or reversetranscriptases having polymerase activity may be isolated from theirnatural viral or bacterial sources according to standard procedures forisolating and purifying natural proteins that are well-known to one ofordinary skill in the art (see, e.g., Houts, G. E., et al., J. Virol.29:517 (1979)). In addition, such polymerases/reverse transcriptases maybe prepared by routine recombinant DNA techniques well know to thoseskilled in the art (see, e.g., Kotewicz, M. L., et al., Nucl. Acids Res.16:265 (1988); U.S. Pat. No. 5,244,797; WO 98/47912; Soltis, D. A., andSkalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376 (1988)).

Recombinant, mutants and other variants of the polymerases describedherein may also be used. Recombinant variants may be particularly usefulin some embodiments described herein. For example, using recombinant DNAtechnology, skilled artisans can generate a polymerase that contains anamino acid epitope that is the target of a polymerase-binding antibody.Alternatively, or in addition, the epitope could bind other moleculessuch as ligands, receptors, haptens and the like. As a result, by addinga particular epitope to several polymerases, it is possible to inhibit arange of different polymerases with a single PBM in the presence of aPBM-binding molecule/complex. In another embodiment, the conditionsunder which a given polymerase is inhibited by a given inhibitor can beoptimized by modifying the inhibitor and/or, through recombinanttechnologies, the polymerase.

Antibodies

PBAs and PBA-binding antibodies may be polyclonal or monoclonal, and maybe prepared by any of a variety of methods (see, e.g., U.S. Pat. No.5,587,287). For example, polyclonal antibodies may be made by immunizingan animal with one or more polypeptides having polymerase activity orportions thereof according to standard techniques (see, e.g., Harlow,E., and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (1988); Kaufman, P. B., etal., In: Handbook of Molecular and Cellular Methods in Biology andMedicine, Boca Raton, Fla.: CRC Press, pp. 468-469 (1995)).Alternatively, anti-polymerase monoclonal antibodies (or fragmentsthereof) may be prepared using hybridoma technology that is well-knownin the art (Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J.Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, NewYork: Elsevier, pp. 563-681 (1981); Kaufman, P. B., et al., In: Handbookof Molecular and Cellular Methods in Biology and Medicine, Boca Raton,Fla.: CRC Press, pp. 444-467 (1995)). Monoclonal PBAs typically have apolymerase association constant of at least about 10⁷ molar⁻¹, althoughantibodies having lower affinities may also be used.

Exemplary antibodies include: Anti-SSIII mAb clone #4; Anti-SSIII mAbclone #2; Primary anti-Tzi antibody clone 8C9; Primary anti-Tzi antibodyclone 3B11.2; Primary anti-Tzi antibody clone 8F3.2; Primary anti-Tziantibody clone 9G2.2; Primary anti-Tzi antibody clone 802.2; Primaryanti-Tzi antibody clone 10F7.3; Primary anti-Tzi mAb clone 1G11.3;anti-ThermoScript mAb DE11; Primary anti-Tzi antibody clone 9G3.3,Primary anti-Tzi antibody clone 6F3.3, anti-RT-41 mAb #5, anti-RT-41 mAb#10, anti-Taq mAb #5 and anti-Taq mAb #10.

It will be appreciated that Fab, F(ab′)₂ and other fragments of theabove-described antibodies may be used in the methods described herein.Such fragments are typically produced by proteolytic cleavage, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments). Antibody fragments may also be produced through theapplication of recombinant DNA technology or through syntheticchemistry.

Formulation of Compositions

Compositions of the invention can include, in addition to a PBM and aPBM-binding molecule/complex, one or more polymerases. Some suchcompositions include one or more thermostable polymerases in addition toa PBM and a PBM-binding molecule. Any thermostable polymerase, PBM(e.g., PBA) that binds to the polymerase, and PBM-bindingmolecule/complex are suitable for use in the invention, including thosedescribed herein. In methods or compositions involving the use orpresence of polymerase, the polypeptides having polymerase activity areused in the present methods at a final concentration in solution ofabout 0.1-4000 units/ml, about 0.1-1000 units/ml, about 0.1-500units/ml, about 0.1-250 units/ml, about 0.1-100 units/ml, about 0.1-50units/ml, about 0.1-40 units/ml, about 0.1-36 units/ml, about 0.1-34units/ml, about 0.1-32 units/ml, about 0.1-30 units/ml, or about 0.1-20units/ml. In one embodiment, the polypeptides having nucleic acidpolymerase and/or reverse transcriptase activity are used at a finalconcentration in solution of about 20 units/ml. Of course, othersuitable concentrations of reverse transcriptase enzymes and nucleicacid polymerases will be apparent to one of ordinary skill in the art.

Compositions in accord with the invention also can include one or moreDNA modifying enzymes (e.g., ligase, kinase, phosphatase, nuclease,endonuclease, exonuclease, topoisomerase, gyrase, terminaldeoxynucleotidyl transferase), nucleic acid templates, nucleic acidprimers, nucleic acid substrates (e.g., rATP, rCTP, rGTP, rTTP, rUTP,rITP, dATP, dCTP, dGTP, dTTP, dUTP, dITP, ddATP, ddCTP, ddGTP, ddTTP,ddUTP, ddITP, and derivatives thereof, including labeled nucleosides andnucleotides), detectable nucleic acid primers, and combinations thereof.

Compositions of the invention also can include one or more detergents(e.g., TRITON X-100°, Nonidet P-40, Tween 20, Brij 35, sodiumdeoxycholate and sodium dodecylsulfate), enzyme cofactors, buffers(e.g., tris(hydroxymethyl)aminomethane,N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid,N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid),3-(N-morpholino)propanesulfonic acid andN[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid, phosphate salts(such as sodium phosphate (mono- or dibasic) and potassium phosphate),sodium bicarbonate, and sodium acetate. Ammonium sulfate, magnesium salt(e.g., magnesium chloride and magnesium sulfate), manganese salt (e.g.,manganese sulfate) and potassium salts (e.g., potassium chloride) alsomay be included in compositions of the invention. One or more chelatingagents such as ehylenediaminetetraacetate (EDTA) may also be included(e.g., at a concentration of about 0.1 millimolar).

Compositions of the invention generally are at a pH in the range of fromabout 7.5 to about 9.5 (e.g., at a pH of from about 8 to about 9).

The reagents described herein are provided and used in any concentrationsuitable for a given use. Required amounts of primers, cofactors andnucleotide-5′-triphosphates needed for amplification or other reactions,and suitable ranges of each are well known in the art. The amount ofcomplex of polymerase and the inhibitor is generally enough to supply atleast about 1 unit of enzyme per 100 μl of reaction mixture once theinhibitor becomes ineffective. In one embodiment, from about 1 to about16 units of polymerase per 100 μl of reaction mixture are needed forPCR, and depending upon the particular activity of a given enzyme, theamount of complex is readily determined by one skilled in the art. Theamount of inhibitor present in the composition is generally from about0.5 to about 5 moles of inhibitor per mole of DNA polymerase. In oneembodiment, from about 1 to about 3 moles of inhibitor per mole of DNApolymerase is used.

Compositions may be formulated at working concentrations, or insolutions of higher reagent concentrations (e.g., 2×, 2.5×, 5×, 10×,20×, 25×, 50×, 10×, 250×, 500× and 1000×) that may then be dilutedbefore use.

Methods

The invention provides methods involving the use of compositions thatcontain a PBM and a PBM-binding molecule/complex, e.g., to inhibit apolymerase. The compositions described herein are particularly usefulfor nucleic acid synthesis, sequencing, amplification, and cloning. Suchmethods typically involve bringing a polymerase into contact with a PBM(e.g., PBA) and a PBM-binding molecule/complex, where binding of the PBMto the polymerase does not substantially inhibit the polymerase activityof the polymerase, but where binding of the PBM and PBM-bindingmolecule/complex together substantially inhibit the polymerase activityof the polymerase. Methods of the invention may also involve reversinginhibition caused by a PBM and PBM-binding molecule/complex.

Synthesis, Amplification and Sequencing

The compositions described herein are particularly useful in methods forsynthesizing, amplifying and sequencing nucleic acid molecules. Nucleicacid synthesis methods of the invention can be used to make any nucleicacid molecule from DNA or RNA templates, including DNA molecules, RNAmolecules, or hybrid DNA/RNA molecules, and of which may bedouble-stranded or single-stranded. As such, the methods and/orcompositions of the invention may be used in any technique including,but not limited to, primer extension, transcription, amplification, PCR,reverse transcription, sequencing and the like.

Nucleic acid synthesis and amplification methods in which the presentcompositions may be used include PCR® (U.S. Pat. Nos. 4,683,195 and4,683,202), Strand Displacement Amplification (SDA; U.S. Pat. No.5,455,166; EP 0 684 315), Nucleic Acid Sequence-Based Amplification(NASBA; U.S. Pat. No. 5,409,818; EP 0 329 822), and AbortiveTranscription (Published U.S. Patent Application No. 2003/0099950-A1).Nucleic acid sequencing techniques include dideoxy sequencing methodssuch as those disclosed in U.S. Pat. Nos. 4,962,022 and 5,498,523, aswell as more complex PCR-based nucleic acid fingerprinting techniquessuch as Random Amplified Polymorphic DNA (RAPD) analysis (Williams, J.G. K., et al., Nucl. Acids Res. 18(22):6531-6535, 1990), ArbitrarilyPrimed PCR (AP-PCR; Welsh, J., and McClelland, M., Nucl. Acids Res.18(24):7213-7218, 1990), DNA Amplification Fingerprinting (DAF;Caetano-Anollés et al., Bio/Technology 9:553-557, 1991), microsatellitePCR or Directed Amplification of Minisatellite-region DNA (DAMD; Heath,D. D., et al., Nucl. Acids Res. 21(24): 5782-5785, 1993), andAmplification Fragment Length Polymorphism (AFLP) analysis (EP 0 534858; Vos, P., et al., Nucl. Acids Res. 23(21):4407-4414, 1995; Lin, J.J., and Kuo, J., FOCUS 17(2):66-70, 1995).

Nucleic acid amplification methods comprise contacting a nucleic acidmolecule to be amplified with one or more of the compositions describedherein, thus providing a population of amplified copies of the nucleicacid molecule. Nucleic acid sequencing methods comprise contacting thenucleic acid molecule to be sequenced with one or more of thecompositions described herein. According to these methods, amplificationand sequencing of the nucleic acid molecule may be accomplished by anyof the above-described amplification and sequencing techniques. In oneembodiment, amplification and sequencing is performed by PCR. Thepresent amplification and sequencing methods may be used foramplification and sequencing of nucleic acid molecules between about 0.5and 7 kb, 0.5-5 kb, 1-5 kb, 1-3 kb or 1-2 kb.

Nucleic acid synthesis, amplification and sequencing methods typicallyinvolve contacting a template nucleic acid with a composition comprisinga polymerase (e.g., thermostable polymerase), a PBM, a PBM-bindingmolecule/complex, and nucleotide substrates under conditions where thepolymerase activity of the polymerase is inhibited. Such reactionmixtures typically include a nucleic acid primer as well. Such nucleicacid synthesis, amplification and sequencing methods typically involvebringing such reaction mixtures to a condition (e.g., highertemperature) that is sufficient to reverse inhibition of the polymerasebut that does not substantially reduce the polymerase's polymeraseactivity. For example, such inhibition reversal may be accomplished byshifting a reaction mixture to a temperature up to 40° C., 50° C., 55°C., 60° C., 65° C., 70° C., 75° C., 80° C., 90° C., 95° C. or 99° C.This heating may be sufficient to cause denaturation of the PBM (e.g.,antibody); denaturation of the PBM-binding molecule/complex;dissociation of the PBM from the nucleic acid polymerase; dissociationof the PBM-binding molecule/complex from the PBM; dissociation of thePBM-binding molecule/complex, or a combination of these effects.

Cloning

Methods of cloning nucleic acids also are provided. Such cloning methodstypically involve synthesizing or amplifying one or more nucleic acidmolecules using a polymerase; incubating the synthesized nucleic acidswith a PBM and a PBM-binding molecule/complex, such that binding of thePBM to the polymerase does not substantially inhibit the polymeraseactivity of the polymerase, but such that binding of the PBM andPBM-binding molecule/complex together substantially inhibit thepolymerase activity of the polymerase; and inserting the amplified orsynthesized nucleic acid molecules into one or more host cells.

The invention provides cloning methods that involve the use of a PBM andPBM-binding molecule, whereby residual polymerase activity remaining inthe reaction mixture after nucleic acid amplification or synthesis isinactivated or inhibited. By the methods described herein, amplified,synthesized or digested nucleic acid molecules may be quickly andefficiently ligated (using ligases, topoisomerases, etc.) into cloningvectors, and these vectors then inserted into host cells.

One exemplary cloning method involves: (a) amplifying or synthesizingone more nucleic acid molecules in the presence of one or morepolymerases to produce amplified nucleic acid molecules; and (b)incubating the nucleic acid molecules with a PBM and PBM-bindingmolecule under conditions sufficient to inhibit or inactivate thepolymerase activity of the polymerase.

In one embodiment, the amplified nucleic acid fragments may be cloned(ligated) directly into one or more vectors to produce one or moregenetic constructs. The genetic constructs then may be transformed intoone or more host cells.

In other exemplary cloning methods, amplified molecules cleaved ordigested with one or more restriction enzymes or one or morerecombination proteins as described in more detail below are cloned intoappropriate insertion sites of cloning vectors (see, e.g., Ausubel, F.M., et al., eds., “Current Protocols in Molecular Biology,” New York:John Wiley & Sons, Inc., pp. 3.16.1-3.16.11 (1995)). Restriction enzymesused for cleavage of the amplified molecules may include blunt-endcutters (e.g., SmaI, SspI, ScaI, etc.) and sticky-end cutters (e.g.,HindIII, BamHI, KpnI, etc.). Such cloning methods also may involve theuse of uracil DNA glycosylase ((UDG); see U.S. Pat. No. 5,137,814, whichis incorporated herein by reference in its entirety). Such methodstypically involve: (a) forming a mixture comprising one or more nucleicacid molecules, one or more PBMs and one or more PBM-binding molecules;and (b) ligating the nucleic acid molecules into one or more of theabove-described vectors to form one or more genetic constructs.Analogously, methods suitable for cloning a nucleic acid molecule intoone or more vectors typically involve: (a) forming a mixture comprisingnucleic acid molecules to be cloned, cloning vectors and one or morepolymerase inhibitors; and (b) ligating the nucleic acid molecules intoone or more vectors to form one or more genetic constructs. Anotherexemplary cloning method involves: (a) forming a mixture comprising thenucleic acid molecules to be cloned, one or more PBMs, one or morePBM-binding molecules and one or more restriction endonucleases; and (b)ligating the nucleic acid molecules into one or more of theabove-described vectors to form one or more genetic constructs. Anotherexemplary cloning method involves: (a) forming a mixture comprising thenucleic acid molecules to be cloned, one or more PBMs, one or morePBM-binding molecules, and one or more recombination proteins; and (b)ligating the nucleic acid molecules into one or more of theabove-described vectors to form one or more genetic constructs.

The mixture formed in the steps (a) of the above-described methods mayfurther comprise one or more additional components, including, apolymerase, dNTPs or ddNTPs, one or more buffer salts, and the like. Apolymerase and restriction endonuclease or recombination protein may beadded to the mixture simultaneously, or may be added sequentially, inany order.

The exemplary cloning methods may also comprise one or more additionalsteps, such as the transformation of one or more of the geneticconstructs formed by these methods into host cells.

Target Nucleic Acids

Nucleic acid may be DNA (including cDNA), RNA (including polyadenylatedRNA (polyA+RNA), messenger RNA (mRNA), transfer RNA (tRNA) and ribosomalRNA (rRNA)) or DNA-RNA hybrid molecules, and may be single-stranded ordouble-stranded.

Nucleic acids to be cloned, or to serve as templates for sequencing,synthesis, amplification may be derived from a variety of sources. Forexample, target nucleic acids may be prepared synthetically according tostandard organic chemical synthesis methods that will be familiar to oneof ordinary skill or may be obtained from natural sources, such as avariety of cells, tissues, organs or organisms. Nucleic acids and cDNAlibraries may be obtained commercially, for example from Invitrogen(Carlsbad, Calif.) and other commercial suppliers that will be familiarto the skilled artisan.

A target nucleic acid may also be extracted in some manner to make itavailable for contact with the primers and other reagents. This mayinvolve the removal of unwanted proteins and cellular matter from thespecimen. Various procedures for doing this are known in the art,including those described by Laure et al in The Lancet, pp. 538-540(Sep. 3, 1988), Maniatis et al, Molecular Cloning: A Laboratory Manual,pp. 280-281 (1982), Gross-Belland et al in Eur. J. Biochem., 36, 32(1973) and U.S. Pat. No. 4,965,188. Extraction of DNA from whole bloodor components thereof are described, for example, in EP-A-0 393 744(published Oct. 24, 1990), Bell et al, Proc. Natl. Acad. Sci. USA,78(9), pp. 5759-5763 (1981) and Saiki et al, Bio/Technology, pp.1008-1012 (1985).

Variations

Variations of the compositions and methods described herein may beperformed by one of ordinary skill in the art. For example, in any ofthe described methods, a PBM and a PBM-binding molecule/complex may beadded to a nucleic acid synthesis reaction mixture together orseparately. As another example, molecules that comprise a PBA-bindingcomplex may be added to a nucleic acid synthesis reaction mixturetogether or separately. As yet another example, polymerase inhibitioncompositions or methods may include or involve two or more PBMs (one ormore of which can be an antibody (i.e., PBA)), two or more PBM-bindingmolecules/complexes (one or more of which can be or include anantibody), and/or two or more polymerases.

It will be readily apparent to one of ordinary skill in the art thatother suitable modifications and adaptations to the methods andapplications described herein are obvious and may be made withoutdeparting from the scope of the embodiments described herein. Having nowdescribed the present embodiments in detail, the same will be moreclearly understood by reference to the following examples, which areincluded herewith for purposes of illustration only and are not intendedto be limiting.

EXAMPLES Example 1 Multicomponent Inhibition of Superscript III ReverseTranscriptase and Tzi DNA-Dependent DNA Polymerase

Introduction

This example demonstrates that multicomponent inhibitors are effectiveat inhibiting nucleic acid polymerase activity. Such inhibition may bepracticed on any polymerase. In the following example, inhibitors wereidentified for inhibiting an RNA-dependent DNA polymerase (reversetranscriptase) and a DNA-dependent DNA polymerase. The degree ofinhibition could be modulated by selection of different polymerasebinding antibodies as the first molecule, and different antibody bindingmolecules or complexes of molecules as the second component of theinhibitor. Inhibition of a thermostable polymerase was reversed byheating or dilution.

Materials and Methods

Antibody production: Recombinant SSIII, Tzi, Rt41A and Taq DNApolymerases were produced and purified (Invitrogen) and used to raisemonoclonal mouse anti-SSIII, anti-Tzi, anti-Rt41A, and anti-Taqantibodies by commercial antibody producers (Chemicon International,Temecula, Calif. and ProSci Incorporated, Poway Calif.). All antibodieswere verified by the manufacturer to be antigen specific by ELISA.Monoclonal antibodies were purified from hybridoma supernatants orascites fluid using standard protein G chromatography methods.

SuperScript III unit assay: The activity of SSIII with and withoutinhibitors was measured using the MMLV reverse transcriptase unit assay(Invitrogen SOP 30573.SOP) with slight modification. Activity wasmeasured in 25 μl total volume of assay buffer (50 mM Tris-HCl pH8.3, 75mM KCl, 6 mM MgCl₂, 1 mM DTT, 0.5 mM dTTP, 1 mM poly(A)-0.6 mM d(T)₂₅,˜20 μCu/ml [α-³²P]dTTP) containing 1 U of SSIII and varying amounts ofinhibitory components. Incubations were performed at indicatedtemperatures for 15 minutes, and terminated by spotting 20 μl of eachreaction onto Whatman GF/C fiber filters. Unincorporated label wasremoved by TCA washes, and incorporation assessed by liquidscintillation.

Tzi, Rt41A, and Taq unit assay: The activity of Tzi, RT41A, or Taq withand without inhibitors was measured using the Taq/Tsp unit assay(Invitrogen SOP 30382.SOP), with slight modification. Activity wasmeasured in 25 ul total volume in assay buffer (25 mM TAPS pH 9.3, 2 mMMgCl₂, 50 mM KCl, 1 mM DTT, 0,2 mM dNTP mix, 2.5 μg nicked salmon testesDNA, and 21 μCi/ml [α-³²P]dCTP), containing 0.25 U Tzi, RT-41, or Taq,and varying amounts of inhibitory components. Incubations were performedat indicated temperatures for 15 minutes, and terminated by spotting 20μl of each reaction onto Whatman GF/C fiber filters. Unincorporatedlabel was removed by TCA washes, and incorporation assessed by liquidscintillation.

Inhibition of Reverse Transcriptase Activity by MulticomponentInhibitors

To determine if SuperScript III (SSIII) can be inhibited by antibodies,SSIII activity was measured by unit assay in the presence and absence ofmouse anti-SSIII monoclonal antibodies (mAb). Anti-SSIII clones #2 and#4 did not directly inhibit SSIII activity, either alone or incombination, even to 10 fold molar excess of mAb to SSIII (FIG. 1).However, the addition of goat-anti-mouse-IgG-horse radish peroxidase(anti-IgG-HRP) in combination with anti-SSIIII clones #4 and #2 stronglyinhibited SSIII activity (FIG. 1). This inhibition was specific toanti-SSIII mAb components, as SSIII activity was inhibited byanti-IgG-HRP in combination with any of an additional four independentanti-SSIII mAbs, but not with an anti-ThermoScript mAb (DE11) (FIG. 2).Denaturing the anti-SSIII mAbs before adding anti-IgG-HRP prevented themajority of the SSIII inhibition (FIG. 1), as did titrating the amountof anti-IgG-HRP (FIG. 2). Furthermore, anti-IgG-HRP by itself producedlittle inhibition of SSIII activity (data not shown). Taken together,these results indicate that both components of the inhibitory complex(the SSIII-specific mAb and the inhibitory anti-IgG mAb) must be presentfor strong inhibition of SSIII activity.

To determine if components other than anti-IgG-HRP could effectivelyinhibit SSIII activity in conjunction with anti-SSIII, a variety ofmolecules that interact with antibodies were screened for the ability toinhibit SSIII in the presence and absence of anti-SSIII. The lectinConcavalin A, AlexaFluor 488 conjugated streptococcal antibody-bindingprotein G, anti-IgG, and the anti-IgG-biotin+streptavidin complex allproduced little or no inhibition of SSIII activity by themselves (FIG.3, and data not shown for anti-IgG). However, in conjunction withanti-SSIII, protein G-AlexaFluor 488, anti-IgG, andanti-IgG-biotin+streptavidin each partially inhibited SSIII, whileanti-IgG+Protein G-AlexaFluor 488 very strongly inhibited SSIII (FIG.3). These results indicate that a variety of antibody-interactingmolecules can couple with the anti-SSIII to effect SSIII inhibition.

To determine if inhibition of SSIII by the multi-component complex isreversible, SSIII unit-activity was measured at both room temperatureand at a temperature where the antibody should be destabilized/denatured(55°). In agreement with previous experiments, SSIII was stronglyinhibited by both anti-IgG-HRP and anti-IgG+Protein G-AlexaFluor 488(Gmix) at room temperature in the presence of anti-SSIII but notanti-ThermoScript (DE11) (FIG. 4). As expected, the SSIII inhibitionseen at room temperature was strongly reversed at 55°. These datademonstrate that these SSIII-inhibitor formulations have the potentialto be useful, reversible, inhibitors of RT activity at room temperature.Inhibition of Tzi, Rt41A and Taq polymerase activity by multicomponentinhibitors

To determine if the principle of multi-component inhibitors can beapplied to enzymes other than SSIII, inhibition of the thermostable DNApolymerases Tzi, RT41 and Taq was assayed in the presence and absence ofmulti-component inhibitors. Each of six primary mouse anti-Tzi mAbsfailed to directly inhibit Tzi by themselves, as did Gmix by itself(FIG. 5). However, in combination with Gmix, each of the six anti-TzimAbs produced moderate to strong inhibition of Tzi (FIG. 5).Furthermore, as seen with SSIII multicomponent inhibitors, theinhibition of Tzi at lower temperatures was significantly reversed atelevated temperatures (FIG. 6). Multi-component inhibitors have thusbeen demonstrated to be effective and reversible inhibitors of reversetranscriptases and DNA polymerase, and are likely to be broadlyapplicable inhibitors of many classes of enzymes.

To determine the effect of the molar ratio of anti-Tzi monoclonalantibody on Tzi polymerase activity, the Tzi unit assay was performedwith no anti-Tzi monoclonal antibody, or in the presence of increasingmolar ratios of anti-Tzi to Tzi (between 1:256 and 8:1). Tzi unitactivity was measured at 37 C in the presence of increasingconcentrations of anti-Tzi mAbs 9G3.3+6F3.2, either with or without asecondary inhibitor mixture comprising a 4:1 molar ratiorabbit-anti-mouse IgG:Tzi and 4:1 molar ratio protein G:Tzi. At a 1:1molar ratio of anti-Tzi mAbs:Tzi, in the absence of secondaryinhibitors, Tzi activity was potentiated, whereas in the presence ofsecondary inhibitors, Tzi activity was maximally inhibited (FIG. 7). Tziinhibition began to be lost above molar ratios of anti-Tzi mAB:Tzi of2:1, most likely due to titration of the secondary inhibitors by theexcess of Tzi. Similar results to the above were also obtained withRT-41 (FIG. 8) and Taq (FIG. 9) DNA polymerases using anti-Rt41A andanti-Taq monoclonal antibodies with rabbit-anti-mouse-IgG secondaryantibody.

To determine the effect of the molar ratio of rabbit-anti-mouse-IgG onTzi polymerase activity, the Tzi unit assay was performed in thepresence of increasing concentrations of rabbit-anti-mouse-IgG (between1:16 and 8:1) either with or without a 1:1 molar ratio of anti-Tzi mAbs(9G3.3+6F3.2):Tzi. Molar ratios of rabbit-anti-mouse-IgG:Tzi as low as4:1 maximally inhibited Tzi activity in the presence but not the absenceof anti-Tzi mAbs (FIG. 10).

To determine the effect of preincubation temperature on Tzi activity,Tzi alone or (Tzi+anti-Tzi mAbs+rabbit-anti-mouse IgG) was preincubatedat various temperatures for 2 minutes prior to performing the Tzi unitassay. Tzi activity was measured at 37° C. after the preincubation, andnearly full activity was regained after as little as 2 minutespreincubation at 94° C. (FIG. 11).

The multicomponent inhibitor methods described above also improved thespecificity and yield of specific product using PCR.

Antibodies suitable for use in the present invention which bind tonucleic acid polymerases and which do not substantially inhibit thepolymerase activity may be identified using the methods described above.Similarly, antibody-binding molecules or complexes of molecules whichbind to the antibody and substantially inhibit the polymerase activitymay also be identified using the methods described above. Thus, any suchantibody which does not substantially inhibit, and any antibody-bindingmolecule or complex of molecules which substantially inhibits polymeraseactivity are within the scope of the present invention.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. A composition comprising: (a) a nucleic acid polymerase; (b) apolymerase-binding molecule (PBM) that binds to said polymerase and doesnot substantially inhibit the polymerase activity of said polymerase;and (c) a PBM-binding molecule or complex or molecules that binds tosaid PBM, wherein binding of the PBM and PBM-binding molecule or complextogether substantially inhibits the polymerase activity of saidpolymerase.
 2. The composition of claim 1, wherein said PBM is anantibody (PBA).
 3. The composition of claim 2, wherein said antibody isa monoclonal antibody.
 4. The composition of claim 1, wherein saidPBM-binding molecule/complex is selected from the group consisting of:an antibody; protein G; protein A; a derivatized antibody; a derivatizedprotein G; a derivatized protein A; IgG and a derivatized protein G;sir22; sibA; and a complex comprising any one of the foregoing.
 5. Thecomposition of claim 2, wherein said derivatized PBM-binding molecule orcomplex of molecules comprises a moiety selected from the groupconsisting of: a detectable label; a protein; and a polymer.
 6. Thecomposition of claim 5, wherein said detectable label is selected fromthe group consisting of: rhodamine; biotin; fluorescein; horseradishperoxidase; alkaline phosphatase; and AlexaFluor488.
 7. The compositionof claim 5, wherein said protein is selected from the group consistingof: horseradish peroxidase; alkaline phosphatase; and albumin.
 8. Thecomposition of claim 5, wherein said polymer is selected from the groupconsisting of: a polyethylene glycol; a polyoxyethylene; apolyoxypropylene; and a polyoxyethylene/polyoxyethylene copolymer. 9.The composition of claim 2, wherein said antibody is selected from thegroup consisting of (a) IgA; (b) IgG; (c) IgM; (d) IgD; (e) IgE; (f)IgY; (g) a fragment of any of (a)-(f); (h) a derivative of any of(a)-(f); (i) a derivatized fragment of any of (a)-(h); and (j) a complexof any of (a)-(i).
 10. The composition of claim 2, wherein saidPBM-binding molecule/complex is a derivatized or underivatized:monoclonal antibody, polyclonal antibody, Fc antibody fragment, chimericantibody or recombinant antibody.
 11. The composition of claim 10,wherein said derivatized antibody is derivatized by attachment of amoiety selected from the group consisting of: a detectable label; aprotein; and a polymer.
 12. The composition of claim 11, wherein saiddetectable label is selected from the group consisting of: rhodamine;biotin; fluorescein; horseradish peroxidase; alkaline phosphatase; andAlexaFluor488.
 13. The composition of claim 11, wherein said protein isselected from the group consisting of: horseradish peroxidase; alkalinephosphatase; and albumin.
 14. The composition of claim 11, wherein saidpolymer is selected from the group consisting of: a polyethylene glycol;a polyoxyethylene; a polyoxypropylene; and apolyoxyethylene/polyoxypropylene copolymer.
 15. The composition of claim2, wherein said PBM-binding molecule or complex of molecules is selectedfrom the group consisting of: a goat anti-mouse IgG antibody coupled tohorseradish peroxidase; a complex of a goat anti-mouse IgG antibody andprotein G-horseradish peroxidase; protein G-AlexaFluor488; a goatanti-mouse IgG antibody; a complex of streptavidin and an antibody tomouse IgG coupled to biotin; and Protein G.
 16. The composition of claim1, wherein said nucleic acid polymerase is selected from the groupconsisting of: a DNA-dependent DNA polymerase; and an RNA-dependent DNApolymerase.
 17. The composition of claim 16, wherein said polymerase isthermolabile.
 18. The composition of claim 16, wherein said polymeraseis thermostable.
 19. The composition of claim 18, wherein saidpolymerase is selected from the group consisting of: Thermus aquaticus(Taq); Thermus thermophilus (Tth); Thermus filiformis (Tfi); Thermusflavus (Tfl); Pyrococcus furiosus (Pfu); Thermococcus litoralis (Tli);Thermococcus zilligi (Tzi); Thermatoga neopolitana (Tne); Thermatogamaritime (Tma); VENT®; DEEPVENT®; THERMOSCRIPT®; SUPERSCRIPT I®;SUPERSCRIPT II®; SUPERSCRIPT III®; and a mutant of any of the above. 20.The composition of claim 18, wherein said nucleic acid polymerase isrecombinant.
 21. A composition comprising: (a) a thermostable nucleicacid polymerase; (b) a polymerase-binding antibody (PBA) that binds tosaid polymerase and does not substantially inhibit the polymeraseactivity of said polymerase; and (c) a PBA-binding molecule or complexor molecules that binds to said PBA, wherein binding of the PBA andPBA-binding molecule or complex together substantially inhibits thepolymerase activity of said polymerase at a temperature less than about40° C., and wherein binding of the PBA and PBA-binding molecule orcomplex together does not substantially inhibit the polymerase activityof said polymerase at a temperature greater than about 40° C.
 22. Thecomposition of claim 21, wherein said nucleic acid polymerase isselected from the group consisting of: a DNA-dependent DNA polymerase;and an RNA-dependent DNA polymerase.
 23. The composition of claim 22,wherein said nucleic acid polymerase is selected from the groupconsisting of: Thermus aquaticus (Taq); Thermus thermophilus (Tth);Thermus filiformis (Tfi); Thermus flavus (Tfl); Pyrococcus furiosus(Pfu); Thermococcus litoralis (Tli); Thermococcus zilligi (Tzi);Thermatoga neopolitana (Tne); Thermatoga maritime (Tma); VENT®;DEEPVENT®; THERMOSCRIPT®; SUPERSCRIPT I®; SUPERSCRIPT II®; SUPERSCRIPTIII®; and a mutant of any of the above.
 24. The composition of claim 21,wherein said thermostable polymerase is recombinant.
 25. A method ofinhibiting the polymerase activity of a nucleic acid polymerasecomprising contacting said polymerase with a PBM and a PBM-bindingmolecule or complex of molecules, wherein the binding of said PBM doesnot substantially inhibit polymerase activity of said polymerase, andwherein the binding of said PBM and said PBM-binding molecule or complexof molecules together substantially inhibits the polymerase activity ofsaid polymerase.
 26. The method of claim 25, wherein said inhibition isirreversible.
 27. The method of claim 25, wherein said inhibition isreversible by heating to a temperature of at least about 40° C.
 28. Themethod of claim 25, wherein said PBM is a PBA.
 29. The method of claim25, wherein said PBM-binding molecule is an antibody.
 30. The method ofclaim 25, wherein said nucleic acid polymerase is selected from thegroup consisting of: a DNA-dependent DNA polymerase; and anRNA-dependent DNA polymerase.
 31. The method of claim 30, wherein saidnucleic acid polymerase is selected from the group consisting of:Thermus aquaticus (Taq); Thermus thermophilus (Tth); Thermus filiformis(Tfi); Thermus flavus (Tfl); Pyrococcus furiosus (Pfu); Thermococcuslitoralis (Tli); Thermococcus zilligi (Tzi); Thermatoga neopolitana(Tne); Thermatoga maritime (Tma); VENT®, DEEPVENT®; THERMOSCRIPT®;SUPERSCRIPT I®; SUPERSCRIPT II®; SUPERSCRIPT III®; and a mutant of anyof the above.
 32. The method of claim 30, wherein said polymerase isrecombinant.
 33. A method for synthesizing a nucleic acid molecule,comprising: (a) contacting a template nucleic acid with a compositioncomprising a thermostable nucleic acid polymerase, one or morenucleoside and/or deoxynucleoside triphosphates and an inhibitor of saidpolymerase, wherein said inhibitor comprises: (i) a Polymerase bindingmolecule (PBM) that does not substantially inhibit the polymeraseactivity of said polymerase; and (ii) a PBM-binding molecule or complexof molecules, wherein the combination of said PBM and said PBM-bindingmolecule/complex substantially inhibits the polymerase activity of saidpolymerase; and (b) bringing the resulting mixture to a temperaturesufficient to inactivate said inhibitor, but that does not inactivatesaid polymerase.
 34. The method of claim 33, wherein said PBM is aPolymerase Binding Antibody (PBA).
 35. The method of claim 33, whereinsaid PBM-binding molecule is an antibody.
 36. The method of claim 33,wherein said inhibition is irreversible.
 37. The method of claim 33,wherein said inhibition is reversible by heating to a temperature of atleast about 40° C.
 38. The method of claim 33, wherein said nucleic acidpolymerase is selected from the group consisting of: a DNA-dependent DNApolymerase; and an RNA-dependent DNA polymerase.
 39. The method of claim38, wherein said nucleic acid polymerase is selected from the groupconsisting of: Thermus aquaticus (Taq); Thermus thermophilus (Tth);Thermus filiformis (Tfi); Thermus flavus (Tfi); Pyrococcus furiosus(Pfu); Thermococcus litoralis (Tli); Thermococcus zilligi (Tzi);Thermatoga neopolitana (Tne); Thermatoga maritime (Tma); VENT®,DEEPVENT®; THERMOSCRIPT®; SUPERSCRIPT I®; SUPERSCRIPT II®; SUPERSCRIPTIII®; and a mutant of any of the above.
 40. The method of claim 38,wherein said nucleic acid polymerase is recombinant.